effects of advanced selection methods on sperm quality … · effects of advanced selection methods...

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Effects of advanced selection methodson sperm quality and ART outcome: asystematic reviewTamer M. Said1,* and Jolande A. Land2

1Andrology Laboratory and Reproductive Tissue Bank, The Toronto Institute for Reproductive Medicine (ReproMed), 56 AberfoyleCrescent, Toronto, ON, Canada M8X 2W4 2Department of Obstetrics and Gynaecology, University Medical Center Groningen,University of Groningen, Groningen, The Netherlands

Correspondence address. Tel: +1-416-233-8111; Fax: +1-416-233-9180; E-mail: [email protected]

Submitted on April 4, 2011; resubmitted on June 13, 2011; accepted on July 4, 2011

table of contents

† Introduction† Methods† Results

Selection based on sperm surface chargeNon-apoptotic sperm selectionSelection based on sperm membrane maturitySelection based on sperm ultramorphology

† Conclusions

background: Current routine semen preparation techniques do not inclusively target all intrinsic sperm characteristics that may impactthe fertilization potential. In order to address these characteristics, several methods have been recently developed and applied to spermselection. The objective of this study was to systematically review the literature describing these advanced sperm selection methods focusingon their anticipated benefits on sperm quality and assisted reproductive technique (ART) outcome.

methods: Systematic literature review was conducted by means of a Medline literature search. Sperm quality parameters assessedincluded: motility, morphology, viability, DNA integrity, apoptosis and maturity. ART outcomes assessed included: fertilization, embryoquality, pregnancy, abortion and live birth rates.

results: A total of 44 studies were identified describing four advanced sperm selection methods based on: (i) surface charge (electro-phoresis and zeta potential), (ii) apoptosis (magnetic cell sorting and glass wool), (iii) membrane maturity (hyaluronic acid binding) and (iv)ultramorphology (high magnification). Selection of high-quality sperm including improvements in DNA integrity, resulted from the applicationof these methods. Fertilization and pregnancy rates showed improvement following some of the advanced sperm selection techniques.

conclusions: While some of the advanced sperm selection methods are of value in specific clinical ART settings, others are in need offurther evaluation. More clinical studies on safety and efficacy are needed before the implementation of advanced sperm selection methodscould be universally recommended in ART.

Key words: assisted reproductive techniques / hyaluronic acid binding / IMSI / magnetic cell sorting / sperm selection

IntroductionDespite the widespread use of assisted reproductive techniques(ARTs) for many years in the treatment of infertility, live birth ratesremain relatively low and could be improved (Wright et al., 2008).

The possible contribution of sperm selection to this improvementhas yet to be established. Previously, the role of spermatozoa in ferti-lization and embryo development was minimized to being a carrierthat transports DNA to the oocyte. It is now proved that human sper-matozoa play an extensive role that extends even beyond the early

& The Author 2011. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.For Permissions, please email: [email protected]

Human Reproduction Update, Vol.17, No.6 pp. 719–733, 2011

Advanced Access publication on August 25, 2011 doi:10.1093/humupd/dmr032

stages of fertilization to include abnormal embryogenesis leading toimplantation failure (Barroso et al., 2009).

Routine sperm preparation techniques such as density gradient cen-trifugation (DGC) and swim-up are currently used as main componentsof ART procedures. They depend on sedimentation or migrationapproaches to separate spermatozoa (Akerlof et al., 1987). Theseroutine techniques appear to be equally effective in selecting motile, mor-phologically normal sperm (Le Lannou and Blanchard, 1988). However,other sperm characteristics such as apoptosis and apoptosis-like manifes-tations, DNA integrity, membrane maturation and ultrastructure are notdirectly targeted by routine sperm preparation techniques. These charac-teristics could be influenced by sperm selection and concomitantly beimportant determinants of fertility. In support, testicular sperm retrievalwas proved to result in the retrieval of sperm with significantly lowerDNA fragmentation compared with ejaculated sperm in the samepatient (Moskovtsev et al., 2010). The same concept is validated in astudy documenting higher ongoing clinical pregnancy rates followingICSI with testicular sperm compared with ICSI using ejaculated sperm inpatients with high DNA fragmentation (Greco et al., 2005). Therefore,the need for advanced sperm selection methods is clearly evident.

Several advanced sperm selection methods have been developedwith the objective of improving sperm preparation protocols usedduring ART. These methods aim at isolating mature, structurallyintact and non-apoptotic spermatozoa with high DNA integrity.Similar advanced sperm selection methods are highly needed due tothe extensive use of ICSI in the management of infertile couples.While ICSI has revolutionized ART and offered an effective treatmentoption for severe male factor infertility, its application, if using sperma-tozoa with defective DNA, may result in serious consequences for theoffspring (Ji et al., 1997; Aitken et al., 2003; Aitken and De Iuliis, 2007).

The objective of this review is to summarize the relevant literatureon advanced sperm selection methods used in routine ART treatmentand to make recommendations on their clinical application. We willdescribe their feasibility, safety and effects on sperm quality andART outcomes. The limitations of studies describing the effects ofusing advanced sperm selection methods on ART outcomes, includingsmall sample size will be highlighted. Controversies will be discussedand suggestions for further research provided.

MethodsA MEDLINE literature search was conducted using the key words: semenpreparation or sperm preparation or sperm selection in combination withany of the following: ART outcome, ICSI, IUI, IVF, sperm parameter,sperm quality, apoptosis, DNA, motility, morphology and viability. Thesearch was restricted to human studies published from January 1990 toOctober 2010. No language restrictions were applied. Only originalarticles that assessed the effects of advanced sperm selection methodson sperm characteristics and/or ART outcome were included in this sys-tematic review. Cross-referencing was also conducted from the citations inthe relevant articles obtained from the MEDLINE search. Meetingabstracts were not included in this study.

ResultsOur initial literature search resulted in 943 articles. Articles notreporting on sperm quality or ART outcome and those describing

routine sperm preparation methods such as density gradient andswim-up were excluded on the basis of title and abstract content(n ¼ 912). Cross-referencing of relevant articles resulted in theinclusion of an additional 13 articles. Based on the 44 studies included,we identified four advanced sperm selection methods: (i) selectionbased on sperm surface charge (6 studies), (ii) non-apoptotic spermselection (12 studies), (iii) selection based on sperm membranematurity (12 studies) and (iv) selection based on sperm ultramorphol-ogy (14 studies). For each method, the principles, safety and feasibilitywill be described first, and this will be partly based on literature thatwas not included in the systematic search. Table I summarizes thestudies identified in the systematic literature search, describing theeffects of advanced sperm selection methods on sperm quality,while Table II shows a summary of studies describing effects ofadvanced sperm selection methods on ART outcomes, including fer-tilization rates and clinical pregnancy rates. No articles on the use ofadvanced sperm selection methods prior to IUI were identified.

Selection based on sperm surface chargePrinciples, safety and feasibilityAn electrophoresis-based technology (Microfloww CS-10, Nusep Ltd.,Frenchs Forest, Australia) has been developed to separate spermato-zoa based on size and electronegative charge. The sperm sample sol-ution is loaded into the apparatus’ reservoirs and allowed toequilibrate with special buffer for only 5 min prior to application ofan electric field in the form of constant applied current of 75 mAand a variable voltage of 18–21 V. Sorted sperm can then be retrievedfrom the collection chamber (Fig. 1) (Ainsworth et al., 2005). The sizecriterion ensures that only spermatozoa are included, while leukocytesand immature germ cells are excluded. An electronegative surfacecharge indicates that the sperm is normally differentiated and hasCD52 on its surface (Schroter et al., 1999). This could be thereason for the higher quality of spermatozoa selected by electrophor-esis since CD52 expression was found to be correlated with normalsperm morphology and capacitation (Giuliani et al., 2004). The useof electrophoresis in sperm selection may prompt some safety con-cerns due to the reported negative effects on sperm motility (Engel-mann et al., 1988; Ainsworth et al., 2005).

Electrophoretic sperm selection is relatively fast as it requires only5 min of current application in addition to time for loading, removaland dilution of samples (Ainsworth et al., 2005). It does not entailany centrifugation steps; thus, it avoids the generation of reactiveoxygen species (ROS) that usually occur with centrifugation (Aitkenand Clarkson, 1988). Consequently, the technique may be of greatvalue in minimizing oxidative stress to spermatozoa. Also, the tech-nique has the ability to exclude major sources of ROS such as leuko-cytes and immature germ cells. The efficiency of this technique hasbeen proved when applied to oligozoospermic samples, testicularsperm and frozen spermatozoa. Despite the undisputed benefits ofthis technique, the complexity of the separation apparatus used maybe a limiting factor against its daily routine use in Andrology Labora-tories, specifically those with limited resources.

Another method has been developed based on the sperm zetapotential (electrokinetic potential) (Chan et al., 2006), which is theelectric potential between the sperm membrane and its surroundingsmeasuring 216 to 220 mV in mature sperm (Ishijima et al., 1991).

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Table I Summary of studies describing the effects of advanced sperm selection methods on sperm quality, comparingoutcomes for selected and unselected sperm in the study populations.

Sperm selectionapproach

Author(year)

Sperm selectionmethod

Studydesign

Study population Outcomes reported

(i) Selection based onsurface charge

Ainsworth et al.(2005)

Electrophoreticseparation(Microfloww, CS-10)

Prospective,controlled

Healthy donors (n ¼ 31) Motility (NS)

Motion kinetics (NS)

Viability (NS)

Morphology (S)

DNA integrity (S)

Chan et al.(2006)

Zeta potential Prospective,controlled

Males in ART program (n ¼ 8) Motion kinetics (S)

Morphology (S)

DNA integrity/maturity (S)

Kam et al.(2007)


Healthy donors (n ¼ 9) Motility (NS)

Morphology (S)

Fleming et al.(2008)

Electrophoreticseparation(Microfloww, CS-10)


Males in ART program (n ¼ 28) Motility (NS)

DNA integrity (NS)

Razavi et al.(2010)

Zeta potential and HAbinding


Male partner from infertile couples(n ¼ 77)

DNA integrity (S)

Zeta potential superior than HAbinding

(ii) Non-apoptoticsperm selection

Said et al.(2005a, b)

DGC + MACS Prospective,controlled

Healthy donors (n ¼ 15) Motility (S)

Morphology (NS)

Viability (S)

Apoptosis markers (MMP, CP-3,EPS) (S)




Sperm cryosurvival rate (NS)




Apoptosis markers (MMP, CP-3,EPS) (S)

DNA integrity (S)

Oocyte penetration (S)

Chromatin decondensation afterHICSI (NS)




Sperm recovery rate (NS)

Grunewaldet al. (2006)


Healthy donors (n ¼ 10) Apoptosis marker (MMP)following cryopreservation (S)

Aziz et al.(2007)



Morphology, SDI (S)

Apoptosis markers (MMP,CP-3) (S)

Continued

Advanced sperm selection methods 721

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Table I Continued


Author(year)

Sperm selectionmethod

Studydesign

Study population Outcomes reported


Molecular glass wool Prospective,controlled

Healthy donors (n ¼ 42) Apoptosis markers (MMP, CP-3,EPS) (S)

MACS



Healthy donors (n ¼ 76) Apoptosis markers (MMP, CP-3,EPS) (S)

Oocyte penetration (S)



Infertile men with abnormal spermparameters (n ¼ 21)

Motility (S)

Apoptosis markers (MMP, CP-3)(S)

Chromatin decondensation afterHICSI (S)

de VanteryArrighi et al.(2009)


Men undergoing fertility evaluation(n ¼ 17)

Apoptosis markers (EPS, MMP)(S)

Lee et al.(2010)


Men from couple with unexplainedinfertility and two failed IUI(n ¼ 60)

Apoptosis markers (EPS, MMP)(S)

DNA integrity (S)

IAR (S)

(iii) Mature spermselection

Huszar et al.(2003)

HA binding Prospective,controlled

Men undergoing fertility evaluation:Maturity markers (n ¼ 30),acrosomal integrity (n ¼ 5)

Maturity markers (CK, HspA2,aniline blue) (S)

Acrosomal integrity (S)

Cayli et al.(2004)



Maturity markers (CK, HspA2) (S)

Apoptosis markers (CP-3, Bcl) (S)

Morphometry (S)

Jakab et al.(2005)



Chromosomal aneuploidies (S)

Ye et al. (2006) HA binding Prospective Males in IVF program (n ¼ 195) Motility and morphologycorrelated with HA binding (S)

Prinosilovaet al. (2009)



Morphology assessed by strictcriteria (S)

Tarozzi et al.(2009)


Males in IVF program (n ¼ 60) DNA fragmentation (S)

Parmegianiet al. (2010a, b)


Males in ICSI program: DNAfragmentation (n ¼ 20), MSOME(n ¼ 15)

DNA fragmentation (S)

MSOME (S)

(iv) Selection basedon ultramorphology

Garolla et al.(2008)

High magnification(×13 000)


Men with severe oligozoospermia(n ¼ 20) compared withnormozoospermic controls(n ¼ 10)

MMP (S)

DNA fragmentation (S)

Sperm aneuploidies (S)

Franco et al.(2008)

High magnification(×8400)

Prospective Men undergoing fertility evaluation(n ¼ 30)

DNA fragmentation anddenaturation in sperm with normalnucleus versus with large vacuoles(S)

ART, assisted reproductive techniques; Bcl, B cell lymphoma protein; CK, creatine kinase; CP-3, caspase-3; DGC, density gradient centrifugation; EPS, externalized phosphatidylserine;HA, hyaluronic acid; HICSI, hamster oocyte ICSI; HspA2, heat shock protein; IAR, induced acrosome reaction; ICSI, intracytoplasmic sperm injection; IUI, intrauterine insemination;IVF, in vitro fertilization; MACS, magnetic-activated cell sorting; MSOME, motile sperm organelle morphology examination; MMP, mitochondrial membrane potential; NS, no statisticallysignificant difference; S, statistically significant difference; SDI, sperm deformity index.

722 Said and Land

The zeta potential further decreases with capacitation (Della Giovam-paola et al., 2001). The method entails pipetting washed sperm into apositively charged centrifuge tubes, which can be achieved by simplyrotating a tube two or three times in a latex glove. After 1 min, thetube is centrifuged and inverted to remove all the non-adheringsperm and other contaminants. Thereafter, adhering (negativelycharged, mature) sperm can be retrieved by rinsing the tube withserum-supplemented media (Fig. 2).

The zeta method appears to be easy to perform and is inexpensivesince no electrophoresis equipment is required. Zeta processing wassuccessfully applied on cryopreserved-thawed sperm (Kam et al.,2007). In terms of safety, the method does not include the use of high-voltage electricity; however, there are certain limitations that shouldbe noted. Low sperm recovery was noted following the applicationof zeta method, which limits its use for oligozoospermic samples. Inaddition, the zeta method was not tested on testicular/epididymalsperm or in a humid environment that is known to neutralize electricalsurface charges (Chan et al., 2006).

Effects on sperm quality (Table I)Data evaluating electrophoretic sperm selection using Microfloww

showed that the apparatus yields adequate numbers of spermatozoacompared with DGC and results in the isolation of a sperm populationwith significantly improved morphology and lower leukocyte contami-nation compared with DGC and ×3 centrifugation methods (Ains-worth et al., 2005). Sperm DNA analysis revealed that only samplesprocessed by Microfloww had significantly lower damage than that inthe raw ejaculate in contrast to samples processed by DGC. Therewas a decrease in sperm motility consistent with other reportsabout the negative effects of electrophoresis on sperm motility (Engel-mann et al., 1988); nevertheless, sperm motion kinetics were compar-able with samples processed by DGC and ×3 centrifugation methods(Ainsworth et al., 2005). Similarly, the zeta potential selection methodshowed an ability to yield spermatozoa with significantly higher mor-phology, hyperactivation, DNA integrity and maturity, but not motility,compared with control samples processed by DGC (Chan et al., 2006;Kam et al., 2007; Kheirollahi-Kouhestani et al., 2009; Razavi et al.,2010). The lack of improvement in motility does not indicate anegative effect, unlike the Microfloww electrophoretic separation,which showed a decrease in sperm motility following its application(Ainsworth et al., 2005).

Effects on ART outcome (Table II)The first human live birth following electrophoretic sperm selection(Microfloww, CS-10) and ICSI was reported in a couple that hadseven IVF/ICSI cycles characterized by good fertilization but poorembryo cleavage rates, associated with extensive sperm DNAdamage (Ainsworth et al., 2007). Sperm selection by surface chargeresulted in a decrease in DNA fragmentation and a pregnancy.Although this report described the benefit of using Microfloww in asingle case characterized by high sperm DNA fragmentation, a pro-spective study revealed contradicting results. In this case-series, eachsemen sample was split between preparation with the CS-10 andpreparation by standard DGC, and each cohort of oocytes was splitfor insemination (IVF) or injection (ICSI) using either CS-10 orDGC-prepared spermatozoa. Results showed no significant differencebetween the ability of CS-10 and DGC-prepared spermatozoa to

produce fertilization, embryo cleavage and high-quality embryos.The only advantage noted by the authors was that CS-10 was lesstime-consuming than DGC (Fleming et al., 2008).

In order to evaluate a combined DGC/zeta potential sperm selec-tion method, a trial was conducted in couples undergoing ICSI due tomale factor infertility. Half of oocytes retrieved were inseminated withsperm prepared using the combined protocol, while the other half wasinseminated with sperm prepared using DGC only to serve as con-trols. The fertilization rates were significantly higher in couples receiv-ing at least one embryo from the zeta group. No differences wereseen in embryo cleavage or quality (Kheirollahi-Kouhestani et al.,2009). However, actual pregnancy rates could not be evaluatedsince embryo transfers were done with embryos from both treatmentand control group in the same patient.

Non-apoptotic sperm selectionPrinciples, safety and feasibilityThe externalization of phosphatidylserine (PS) to the outer surface ofthe sperm membrane, a feature of early apoptosis, has been used as abasis for selection of non-apoptotic spermatozoa. The externalizationof PS allows for its binding with Annexin-V-conjugated paramagneticmicrobeads, which could be used to label and separate apoptoticspermatozoa using a magnetic-activated cell sorting system (MACS,Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) (Grunewaldet al., 2001). Initially, a heterogeneous sperm cell suspension is incu-bated with Annexin-V-conjugated microbeads, which bind to onlyapoptotic sperm with externalized PS. Thereafter, the bead/spermmixture is allowed to run through the MACS column, which isplaced inside a magnet. The magnetic force will cause the retentionof the cells labeled with microbeads inside the column, while the non-labeled cells will freely flow (Fig. 3) (Manz et al., 1995). Since MACSdoes not have the ability to remove leukocytes or immature germcells, the technique was used in conjunction with DGC to excludethe seminal plasma and other contaminants (Said et al., 2005a, b).

Annexin V magnetic cell separation of non-apoptotic spermatozoais simple, fast, inexpensive and highly specific (Said et al., 2008).However, the technique still requires special laboratory equipment,which may not be feasible or available in all settings. In addition, thecombination of DGC and MACS will involve repeated steps of cen-trifugation and re-suspension, which might be detrimental whenapplied to semen samples characterized by limited sperm counts, aslow sperm recovery may be expected (Said et al., 2008).

Some safety aspects should be considered before using MACS forsperm selection. MACS microbeads are biodegradable and do notaffect cell viability (Miltenyi et al., 1990); however, research is stillneeded to ensure the complete absence of freely floating microbeadsin the non-apoptotic sperm fraction.

In order to avoid the problems associated with freely floatingmicrobeads, another system for selection of non-apoptotic spermato-zoa has been recently described. The system is based on modifyingcommercially available glass wool separation columns (SpermFertil,TranMIT, GmbH, Giessen, Germany) to add the ability of retainingapoptotic sperm. This was achieved by coating the glass wool withAnnexin V that binds to apoptotic sperm. The technique referred toas Annexin V glass wool (annexin V-GW) or molecular glass wool iseasy to use on both fresh and cryopreserved semen samples;


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Table II Summary of studies describing the effects of advanced sperm selection methods on ART outcomes includingfertilization rates and clinical pregnancy rates.


Author (year) Sperm selectionmethod

Study design Population Outcomesreported

(i) Selection basedon surface charge

Ainsworth et al. (2007) Electrophoreticseparation(Microfloww,CS-10)

Case report One infertile couple Fertilization and livebirth

Fleming et al. (2008) Electrophoreticseparation(Microfloww,CS-10)


Couples undergoing IVF (n ¼ 17) orICSI (n ¼ 11), split oocytesinseminated with sperm prepared byCS-10 (n ¼ 197) versus DGC(n ¼ 195)

FR (IVF) 62 versus 69%(NS)

FR (ICSI) 64 versus 52%(NS)

PR NA

Kheirollahi-Kouhestaniet al. (2009)


Couples undergoing ICSI (n ¼ 30),controls (n ¼ 34)

FR 66% versus 52% (S)

PR 54% versus 33%(NS)

PR NA

(ii) Non-apoptoticsperm selection

Dirican et al. (2008) DGC + MACS Prospective,controlled

Study group (n ¼ 122) versus controls(n ¼ 74)

FR 69% versus 70%(NS)

PR 48% versus 37% (S)

(iii) Mature spermselection

Ye et al. (2006) HA binding Prospective Couples undergoing IVF (n ¼ 175) HBA score when FR.50 versus HBA scorewhen FR ≤50%: 75%versus 69% (marginal S)

Nasr-Esfahani et al.(2008)


Couples undergoing ICSI (n ¼ 50),sibling oocytes inseminated with spermprepared by HA versus DGC



Tarozzi et al. (2009) HA binding Prospective Couples undergoing IVF (n ¼ 60) FR when HBA score≥80% versus FR whenHBA score ,80%: 86%versus 87% (NS)

PR when HBA score≥80% versus PR whenHBA score ,80%: 36%versus 32% (NS)

Van Den Bergh et al.(2009)

HA binding Prospective,randomized

Couples undergoing ICSI (n ¼ 44),oocytes injected with HA bound(n ¼ 204) versus non-bound sperm(n ¼ 203)


PR NA

Parmegiani et al.(2010a, b)

HA binding Prospective,randomized

Couples undergoing ICSI (n ¼ 125)versus controls (n ¼ 107)

FR 92 versus 86% (NS)


Parmegiani et al.(2010a, b)

HA binding Retrospective,controlled

Couples undergoing ICSI (n ¼ 293)versus controls (n ¼ 86)


PR 33% verus 22% (NS)

(iv) Selection basedonultramorphology

Bartoov et al. (2002) MSOME Prospective Randomly selected cases undergoingICSI (n ¼ 100)

Normal nucleus byMSOME when FR.60% versus when FR,60%: 32% versus22% (S)

Continued

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Table II Continued


Author (year) Sperm selectionmethod

Study design Population Outcomesreported

Normal nucleus byMSOME when pregnantversus not pregnant34% versus 25% (S)

Bartoov et al. (2003) IMSI Prospective,controlled

Cases undergoing IMSI (n ¼ 50) versuscontrols (n ¼ 50)

FR 63% versus. 64%(NS)


Berkovitz et al. (2005) IMSI Retrospective,case–controlled

Cases undergoing IMSI with spermhaving normal nucleus (n ¼ 38) versuscases with sperm having abnormalnucleus (n ¼ 38)


PR 52% versus18% (S)

Berkovitz et al.(2006a, b)

IMSI Retrospective,case–controlled

Cases undergoing IMSI with spermidentified with no vacuoles (n ¼ 28)versus cases with sperm identified withvacuoles (n ¼ 28)



Berkovitz et al.(2006a, b)

IMSI Prospective,controlled

Cases undergoing IMSI (n ¼ 80) versuscontrols (n ¼ 80)



Hazout et al. (2006) IMSI Prospective,controlled

Cases undergoing IMSI (n ¼ 125), oneadditional routine ICSI cycle wasincluded from each as a control

FR NA


Check et al. (2007) IMSI Case report One case of globozoospermia Fertilization

Antinori et al. (2008) IMSI Prospective,randomized

Couples with male partner diagnosedwith OAT: IMSI (n ¼ 227) versusroutine ICSI (n ¼ 219)

FR NA


Vanderzwalmen et al.(2008)

IMSI Prospective Couples with male factor infertility(n ¼ 67), sperm with no nuclearvacuoles versus sperm with largenuclear vacuoles

FR NA

PR NA

Mauri et al. (2010) IMSI Prospective,controlled

Couples with male factor and/or twoprevious failed IVF or ICSI siblingoocytes injected by ICSI (n ¼ 172)versus IMSI (n ¼ 159)


Gianaroli et al. (2008) ICSI usingpolarizationmicroscopy

Prospective,randomized

Couples undergoing ICSI underpolarizing lenses (n ¼ 112) versuscontrols undergoing routine ICSI(n ¼ 119)



Gianaroli et al. (2010) ICSI usingpolarizationmicroscopy

Prospective,randomized

Men with severe male factor infertilityundergoing ICSI under polarizinglenses (n ¼ 71)


Outcomes of acrosome-reactedsperm compared withnon-acrosome-reacted sperm


Results are presented as comparison between infertile couples in whom advanced sperm selection methods were performed (study group) versus infertile couples in whom no specificsperm selection was done (controls), unless otherwise noted.ART, assisted reproductive techniques; DGC, density gradient centrifugation; FR, fertilization rate; HA, hyaluronic acid; HBA, hyaluronic acid binding assay; ICSI, intracytoplasmicsperm injection; IMSI, intracytoplasmic morphologically selected sperm injection; IVF, in vitro fertilization; MACS, magnetic-activated cell sorting; MSOME, motile sperm organellemorphology examination; NA, not available; NS, non-significant; OAT, oligoasthenoteratozoospermia; S, statistically significant.


nevertheless, more studies are still needed to validate its efficiency andstandardization (Grunewald et al., 2007).

Effects on sperm quality (Table I)In fresh and cryopreserved-thawed samples of healthy donors, MACSused in combination with DGC selected spermatozoa with highermotility and mitochondrial membrane potential but lower activecaspase-3 and PS externalization compared with sperm selected byDGC only (Said et al., 2005a, b; Grunewald et al., 2006, 2008).Results from a study on samples from healthy donors showed thatthe average number of spermatozoa lost during the combinedapproach was an additional 15% compared with samples prepared

by DGC only (Said et al., 2006a, b). Similar to MACS, annexinV-GW showed an ability to select spermatozoa with lower caspaseactivation and higher mitochondrial membrane potential (Grunewaldet al., 2007). Significantly higher motility, mitochondrial membranepotential, survival following 24 h of incubation and normal sperm mor-phology as assessed by the strict criteria and the sperm deformityindex were seen in fractions prepared by DGC + MACS comparedwith fractions prepared by DGC only (Aziz et al., 2007; de VanteryArrighi et al., 2009).

In healthy donors and men with unexplained infertility, spermatozoaprepared by MACS after DGC had 30% lower rate of DNA damagecompared with those prepared by only DGC (Said et al., 2006a, b;Lee et al., 2010). Since DGC by itself was reported to select sperma-tozoa with 50% lower DNA damage (Donnelly et al., 2000), theresults evaluating MACS indicate that a further decrease in percentageof DNA-damaged sperm is possible, which can be of benefit in somepatients that present with a high percentage of DNA-fragmentedsperm. The combination of DGC and MACS also appears to be ofbenefit in enhancing sperm cryosurvival rates. Non-apoptotic sperma-tozoa selected by DGC and MACS displayed significantly higher cryo-survival rates following cryopreservation-thawing compared withsamples prepared by DGC only (Said et al., 2005a, b).

Hamster oocytes were used to evaluate oocyte penetration poten-tial and chromatin decondensation of non-apoptotic sperm separatedby DGC and MACS. When selected from healthy donors, non-apoptotic sperm exhibited significantly higher oocyte penetrationpotential than controls prepared by DGC (Said et al., 2006a, b).Sperm chromatin decondensation up to 18 h following hamsteroocyte ICSI in fractions selected by DGC and MACS from samplesof healthy donors was comparable with controls prepared by DGConly (Said et al., 2006a, b). However, when using samples from infer-tile men with abnormal sperm parameters, sperm chromatin

Figure 1 Schematic diagram for the electrophoretic system used for sperm selection. (A) Competent spermatozoa move in the applied electricfield, and cell contaminants are excluded based on size. (B) Graphic representation of the system configuration including restriction and separationmembranes, buffer flows and sample inoculation and collection locations. (Ainsworth et al. 2005, by permission of Oxford University Press.).

Figure 2 Diagram for sperm selection using zeta potential. Spermsuspension is pipetted into a positively charged tube. Negativelycharged mature sperm (A) will adhere to the tube, while non-maturesperm (B) will not adhere and are discarded.

726 Said and Land

decondensation up to 18 h following hamster oocyte ICSI was signifi-cantly higher than that of controls prepared by DGC only (Grunewaldet al., 2009). Samples from infertile patients with abnormal sperm par-ameters show higher levels of apoptosis markers. This may be thereason why the beneficial effects of MACS on sperm chromatindecondensation were noted only in samples from infertile patientsand not in samples from healthy donors.

Effects on ART outcome (Table II)In order to assess the value of using a combination of DGC and MACSin a clinical ART setting, a study was conducted by subjecting semensamples from men with abnormal sperm parameters to preparationby DGC and MACS before undergoing ICSI (Dirican et al., 2008).Outcomes were compared with controls whose semen sampleswere prepared by DGC only. There were no differences in fertilizationrates between the study group and the control group. On the otherhand, higher embryo cleavage rates and clinical pregnancy rateswere seen in the study groups compared with controls (Dirican

et al., 2008). Recently, case reports described the use of MACSprior to an ICSI cycle that resulted in the birth of healthy children(Polak de Fried and Denaday, 2010; Rawe et al., 2010). The casesreported had a history of failure of fertilization and poor embryodevelopment following routine ICSI, and the male partners had a sig-nificant level of sperm DNA fragmentation and activated caspase-3.The findings of these cases support the benefit of integrating MACSinto the ART protocol in cases with a high incidence of apoptotic,DNA-fragmented sperm.

Selection based on sperm membranematurityPrinciples, safety and feasibilityThe formation of hyaluronic-acid (HA)-binding sites on the spermplasma membrane is one of the signs of sperm maturity that hasbeen used as a basis for sperm selection (Huszar et al., 1997). Adevice called a PICSI dish (MidAtlantic Diagnostics Inc., Mt Laurel,NJ, USA) has been developed by adding four marked spots of

Figure 3 Schematic diagram of MiniMACSTM magnetic cell separation column. (A) The column that contains steel spheres is placed inside an exter-nal magnet. (B) The non-labeled cells flow through the column to be collected. (C) The immuno-magnetically labeled cells remain attached to themagnetized spheres and are retained inside the column.

Figure 4 Sperm selection using PICSI dishes. (A) A sperm drop is placed at the periphery of a HA drop, mature sperm binds to the HA-spot, whileimmature sperm moves freely. (B) Bound sperm could be picked up with the ICSI pipette. (Jakab et al., 2005, with permission from Elsevier.).


immobilized HA in a Falcon Petri dish. One drop of washed sperma-tozoa is placed at the edge of the HA spot and the HA-bound sper-matozoa are collected after 15 min in an ICSI pipette and used forinjection (Fig. 4) (Jakab et al., 2005). This method of sperm selectionis highly specific and has minimal safety concerns (Huszar et al., 2003).While it is possible that HA molecules will be carried with the selectedsperm within the ICSI pipette, HA is a normal component in cervicalmucus, cumulus cells and follicular fluid (Cayli et al., 2003). To date, noadverse reactions on fertilization or embryo development have beenreported following the use of HA-selected spermatozoa in clinicalIVF settings (Huszar et al., 2007). However, it is important to notethat this added step will eventually require embryologists to investmore time into preparatory steps since sperm binding by itself maytake up to 30 min, which could be a challenge when injecting a largenumber of oocytes.

Effects on sperm quality (Table I)Spermatozoa selected via HA binding display manifestations of matur-ity as defined by creatine kinase, heat shock-related protein 2 (HspA2)and aniline blue staining. HA-selected spermatozoa were also ident-ified as viable, with non-reacted acrosomes, lower caspase-3 acti-vation and positive correlation with motility (Huszar et al., 2003;Cayli et al., 2004; Ye et al., 2006). HA-bound spermatozoa havealso displayed significantly less DNA fragmentation compared withspermatozoa prepared by DGC and spermatozoa in unprocessedsemen (Tarozzi et al., 2009). The likelihood of improving a spermpopulation to the level where it includes .14% morphologicallynormal sperm was estimated to increase 3-fold following selectionby HA binding (Prinosilova et al., 2009).

A correlation exists between sperm maturity and the frequency ofchromosomal aneuploidies as shown by the decreased expression ofHspA2 as a common factor underlying both sperm immaturity andaneuploidy (Kovanci et al., 2001). Thus, it has been suggested thatHA-binding could be used to select mature sperm with low frequencyof chromosomal abnormalities to reduce the risks for genetic compli-cations following ICSI associated with the use of abnormal spermato-zoa. In semen samples from men undergoing fertility assessment,HA-bound spermatozoa had a significantly lower frequency of autoso-mal disomy, diploidy and sex chromosome disomy compared withspermatozoa not selected by HA binding (Jakab et al., 2005).

Effects on ART outcome (Table II)The proportion of sperm capable of binding to HA had no correlationwith fertilization, cleavage, good-quality embryos, miscarriage andpregnancy rates in couples undergoing IVF (Tarozzi et al., 2009). Con-sistently, patients with clinical pregnancies had a percentage ofHA-bound sperm that was comparable to those without pregnancy(Tarozzi et al., 2009). Only a weak correlation between the pro-portion of HA-bound sperm and fertilization rates following IVF wasonce demonstrated (Ye et al., 2006). Currently there are no thresholdvalues for sperm HA binding that could predict IVF outcome, whichlimits the value of estimating the proportion of HA-bound sperm inpredicting IVF outcome (Nijs et al., 2010).

Few studies have documented the use of HA-selected sperm inclinical ART settings. In comparison with routine sperm preparationtechniques, spermatozoa selected by HA binding resulted in signifi-cantly higher fertilization rates following ICSI, while pregnancy rates

were only slightly increased (Nasr-Esfahani et al., 2008). The positiveeffects of HA-bound sperm were associated with lower DNA frag-mentation, which suggests that these effects may be attributed atleast in part to selecting spermatozoa with higher DNA integrity(Nasr-Esfahani et al., 2008). In different studies, HA-bound spermato-zoa used for ICSI resulted in significantly higher embryo quality andcleavage rates but not fertilization or pregnancy rates comparedwith spermatozoa conventionally selected (Parmegiani et al., 2010a,b).

In a randomized study, the effects of HA-selected sperm on embryodevelopment as measured by the zygote score (Z-score) wereassessed. Sibling mature oocytes were injected in a randomizedmanner, with either HA-bound or HA-non-bound spermatozoa; nodifferences were found in fertilization rates or Z-scores betweenboth group (Van Den Bergh et al., 2009). However, it has beenargued that the use of non-HA-bound sperm in ICSI may result inrisks for the conceptus since such sperm is documented to have ahigher incidence of aneuploidies and DNA fragmentation (Parmegianiet al., 2009).

Selection based on sperm ultramorphologyPrinciples, safety and feasibilitySperm morphology has been described as one of the major determi-nants of male in vivo and in vitro fertility (Kruger and Coetzee, 1999;Van Waart et al., 2001; van der Merwe et al., 2005). However, ithas been debated that morphology evaluated on random stainedcells from the ejaculate at ×1000 magnification is of limited valueduring ICSI where sperm is selected unstained at ×400 magnification(Bartoov et al., 2002). Alternatively, a new sperm selection methodhas been developed based on the inclusion of only normal spermassessed using real-time motile sperm organelle morphology examin-ation (MSOME) at a magnification of ×6300 (Bartoov et al., 2002).During MSOME, a micro-droplet of motile sperm suspension pre-pared by a routine sperm preparation technique is examined underoil immersion, with an inverted light microscope fitted with high-power Nomarski optics with digital enhancement.

MSOME assesses five sperm organelles (acrosome, postacrosomallamina, neck, tail and mitochondria) that can be classified as eithernormal or abnormal. The sixth organelle (the nucleus) is evaluated forboth shape and chromatin content (vacuolar area) (Fig. 5). Among thesix organelles, the sperm nucleus appears to be the most important ininfluencing ART outcome (Bartoov et al., 2002). Subsequently, a modi-fication of ICSI termed intracytoplasmic morphologically selected sperminjection (IMSI) has been developed (Bartoov et al., 2003). Thisapproach is of particular benefit when used in situations where identifi-cation of specific sperm organelles is required, such as the acrosomalcomponents in cases of globozoospermia (Check et al., 2007).

MSOME followed by IMSI is an elaborate procedure that involvesprolonged sperm manipulation, adding significantly to the routineICSI processing times. It was reported that it could take up to 5 hto perform (Berkovitz et al., 2005). It also requires special instrumen-tation with considerable expense. The subjectivity of the sperm ultra-morphology assessment may be another limiting factor that preventsits widespread use. Classification of normal sperm ultramorphologywill depend on the technician’s training and experience. Whileintra-observer variability was limited during MSOME (Bartoov et al.,

728 Said and Land

2002), the technique will always require a high level of technical exper-tise and inter-observer reproducibility.

A different optical system has been developed to identify the spermbirefringence, which occurs in mature forms due to the presence ofsubacrosomal protein filaments that are longitudinally oriented(Baccetti, 2004). Sperm birefringence can be evaluated using aninverted microscope equipped with polarizing and analyzing lenses,which allows the selection of birefringent, acrosome-reacted sperma-tozoa during ICSI without negatively impacting sperm motility or viabi-lity (Gianaroli et al., 2008). Birefringent spermatozoa can be selectedfor microinjection and these are thought to present with higherquality as the proportion of birefringent sperm has a significant positivecorrelation with other sperm parameters such as concentration, moti-lity and viability (Gianaroli et al., 2008). As for MSOME and IMSI, theselection of spermatozoa using polarizing microscopy will requireadditional instrumentation, time and technical expertise.

Confocal light absorption and scattering spectroscopic (CLASS)microscopy is an optical system capable of evaluating single subcellularorganelles without resulting in cell destruction (Itzkan et al., 2007).When applied to human sperm, CLASS can be used to visualizesperm organelles including the chromatin. Subsequently, this approachmay be used in the future to select spermatozoa with intact chromatinprior to ICSI. The extremely limited time of sperm exposure to light,estimated as 1 second, may favor the occurrence of fewer side effects(Sakkas and Alvarez, 2010). No published reports regarding theimpact of using CLASS on sperm quality and ART outcome are cur-rently available.

Effects on sperm quality (Table I)Immotile spermatozoa identified as normal with no nuclear vacuolesusing high magnification microscopy (×13 0 00) have significantlyhigher mitochondrial membrane potential and lower DNA fragmenta-tion and aneuploidies compared with unselected sperm (Garolla et al.,2008). Consistently, the presence of large nuclear vacuoles (≥50% ofsperm nuclear area) is associated with significantly higher DNA frag-mentation and denaturation compared with spermatozoa with anormal nucleus (Franco et al., 2008).

Effects on ART outcome (Table II)In a feasibility study, assessment by MSOME of spermatozoa remainingfrom a pool used in routine ICSI revealed that the incidence of mor-phologically normal sperm has a significant correlation with fertilizationrates (Bartoov et al., 2002). Additionally, a threshold of 2.5% normalmorphology as assessed by MSOME was highly predictive of fertiliza-tion rates following ICSI (sensitivity 81%, specificity 78%) (Bartoovet al., 2002). Clinical outcomes such as pregnancy and live birthrates were significantly higher following IMSI than routine ICSI, butno differences were noted as regards to fertilization and cleavagerates and embryo morphology (Hazout et al., 2006; Antinori et al.,2008; Mauri et al., 2010). This may suggest that IMSI is not ofbenefit in improving the paternal component in early steps of fertiliza-tion (Mauri et al., 2010). However, later effects on implantation shouldnot be dismissed since higher pregnancy rates and diminished abortionrates were reported in couples that underwent IMSI when comparedwith couples that underwent routine ICSI (Bartoov et al., 2003; Berko-vitz et al., 2006a, b). In support, a recent meta-analysis was conductedby comparing results from 357 IMSI cycles versus 349 routine ICSIcycles. Results showed significant improvement with IMSI inpregnancy and abortion rates but not in fertilization rates (SouzaSetti et al., 2010).

Embryos resulting from the microinjection of birefringent spermato-zoa only of men with severe male factor infertility had significantlyhigher pregnancy rates compared with rates observed after routineICSI (Gianaroli et al., 2008), and lower abortion rates. On the otherhand, fertilization and cleavage rates were comparable with ratesresulting from routine ICSI (Gianaroli et al., 2008). The use of polariz-ing microscopy allows the selection of acrosome-reacted sperm to beused in routine ICSI, which resulted in significantly higher pregnancyrates compared with spermatozoa with non-reacted acrosomes(Gianaroli et al., 2010).

ConclusionsThe evidence supporting the need for specific advanced sperm selec-tion methods to be implemented in ART is considerable. Data indi-cate that even if the best quality spermatozoa are used in ICSI, no

Figure 5 Microgragh depicting sperm morphological attributes as assessed by MSOME. (A) Sperm cell with a morphologically normal nucleus; (B)Small oval nuclear form; (C) Large oval nuclear form; (D) Wide nuclear form; (E) Narrow nuclear form; (F) Regional (acrosomal) nuclear shape dis-order; (G) Oval nuclear shape and large nuclear vacuoles; (H) Abnormal (narrow) nuclear shape + large nuclear vacuoles. (Berkovitz et al., 2005, bypermission of Oxford University Press.).


more than 55% of the selected sperm have normal DNA (Ramoset al., 2004). This further supports the hypothesis that sperm selec-tion methods currently used prior to ART are inadequate and thatother methods need to be considered to ensure that only spermato-zoa with optimum quality are included. Several advanced spermselection methods have been described based on differentapproaches for targeting functionally competent and intact spermato-zoa. This review focused on advanced sperm selection methods thatcould play a role routinely in the clinical ART practice. Othermethods that could be used in unique situations to identify livesperm in cases of specific defects such as total lack of motility(El-Nour et al., 2001; Gerber et al., 2008) were beyond the scopeof this article.

Selection of spermatozoa based on their electronegative surfacecharge, apoptosis markers, membrane maturity (HA binding) andultramorphology has been used to develop technical protocols forsperm isolation. These different approaches aim at including onlymature, non-apoptotic, DNA intact, morphologically normal sperma-tozoa during IVF or ICSI. Published data document the ability of theseadvanced sperm selection methods to isolate spermatozoa with higherquality in terms of motility, morphology, viability, DNA integrity, apop-tosis and maturity markers.

There are contradicting reports regarding whether ART outcomesmay be improved by using advanced sperm selection methods. Elec-trophoretic preparation using Microfloww may be considered forsamples with high DNA damage since it is capable of selectingsperm with intact DNA (Ainsworth et al., 2007). However, the tech-nique did not lead to any improvement in fertilization rates or embryoquality following ICSI (Fleming et al., 2008). Conversely, the zetapotential method was reported in only one study to increase fertiliza-tion, implantation and pregnancy rates (Kheirollahi-Kouhestani et al.,2009). MACS for the selection of non-apoptotic sperm was provedto result in higher embryo cleavage and pregnancy rates but not in fer-tilization or implantation rates (Dirican et al., 2008). The techniqueappears to be of most benefit in samples having significant levels ofDNA damage and apoptosis (Polak de Fried and Denaday, 2010;Rawe et al., 2010).

As regards HA-binding for sperm selection, some reports showincreased fertilization rates with no effects on pregnancy rates, whileothers report positive effects on embryo development and pregnancyrates and none on fertilization rates (Ye et al., 2006; Nasr-Esfahaniet al., 2008; Tarozzi et al., 2009; Parmegiani et al., 2010a, b). Thesediscrepancies may be due to subtle differences in study designs anddata analysis. A relatively recent randomized, double-blinded, multi-site study reported significantly higher clinical pregnancy rates follow-ing PICSI compared with ICSI. It has also been reported that patientswith lower HBA scores stand to benefit the most from the applicationof PICSI (Worrilow et al., 2009). As regards IMSI, ample evidencedocuments the benefits of the technique on late ART outcomessuch as higher pregnancy and live birth rates, and lower abortionrates but not on earlier outcomes such as fertilization, embryo clea-vage rates and embryo morphology (Bartoov et al., 2003; Berkovitzet al., 2006a, b; Hazout et al., 2006; Antinori et al., 2008; Mauriet al., 2010). Finally, it is important to note that healthy live birthswere reported following the use of spermatozoa selected using elec-trophoretic separation, MACS, HA binding and IMSI. Nevertheless, itis not possible, based on the current published data, to identify which

sperm selection method will result in the highest birth rate of healthyoffspring.

Most of the evidence provided regarding the advantages of usingadvanced sperm selection techniques remains to date preliminary innature. Recent presented studies on MACS showed an improvementin pregnancy rates following IUI with Annexin-V-sorted spermalthough the recovery of motile sperm was considerably diminished(Romany et al., 2010). Also, MACS application did not result in anysignificant benefit in cases with high DNA fragmentation or apoptosis(Alvarez Sedo et al., 2010). Similarly, doubling of ongoing pregnancyrate and halving of abortion rate was achieved following IMSI com-pared with ICSI but yet the numbers reported did not reach statisticalsignificance (Oliveira et al., 2010).

The suggested new methodologies vary in terms of instrumentationand time required. Selection using zeta potential and MACS are fairlyquick and inexpensive. Electrophoretic separation, HA binding andIMSI do require relatively expensive instrumentation and/or consider-able time to perform. All advanced sperm selection methods involvesperm manipulations that are more elaborate and time-consumingthan routine sperm preparation techniques currently used. Further-more, some proposed sperm selection protocols use a combinationof both advanced methods and the routinely used ones. The addedtechnical steps and processing times warrant careful assessment ofsome safety aspects since prolonged sperm exposure to non-physiologic conditions may induce iatrogenic damage (Agarwal et al.,1994). This may result in sperm DNA damage (Twigg et al., 1998)leading to aberrant embryo development and most importantlyabnormalities in the offspring presenting as birth defects or genetic dis-orders (Marchetti and Wyrobek, 2005; Verhofstad et al., 2008). Mostrecently, higher frequency of congenital abnormalities and lower birth-weight was reported following IMSI compared with ICSI (Junca et al.,2010), although the differences were not statistically significant.Further assessment of embryo development and long-term followup for anomalies in the offspring, which are currently lacking, shouldbe conducted to provide assurance regarding the use of advancedsperm selection methods in ART. Additionally, other unwantedeffects such as influencing the sex ratio of the offspring should alsobe evaluated.

In conclusion, the first results following IVF and mostly ICSI after theuse of electrophoretic separation, MACS, HA binding and IMSI areencouraging concerning fertilization and pregnancy rates. Despitethese encouraging results, it should be noted that the numbers ofpatients assessed are limited, and most studies are underpoweredto conclude on differences in pregnancy rates and live births. Moreresearch is needed to identify which infertility cases, if not all, willbenefit from the application of these selection methods. Careshould be taken to investigate safety and efficacy aspects of advancedsperm selection methods before their widespread implementation inART. Animal models could be used initially and when proved safe,there will still be a need for adequately powered randomizedtrials in the human setting on efficacy with long-term follow-up ofchildren.

Authors’ rolesT.M.S. took part in acquisition of data, data analysis and interpretation,drafting of article and final approval of the version to be published. J.L.

730 Said and Land

played a role in drafting of article, critical revision of article, and finalapproval of the version to be published.

FundingNo specific funding was either sought or obtained to support thisstudy.

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